How cells regulate and execute cytokinesis, the final step in cell division, remains one of the major unsolved questions in basic biology. Our long-term goal is the systematic dissection of temporal and spatial control during cytokinesis. It has been challenging to study cytokinesis with traditional methods because it is a rapid process and many key cytokinesis proteins also perform important functions earlier in the cell cycle. This is why small molecules, which act rapidly and with high temporal control, are ideal tools to study cytokinesis. We discovered and characterized several novel small molecule inhibitors of cytokinesis. Using these small molecules as tools, we can now address some important outstanding questions in cytokinesis research. For example, how is actin assembled into a contractile ring structure and how does the ring constrict? How is cytokinesis regulated, both at a detailed and more global level? Based on intriguing preliminary characterizations, we selected three small molecules with different mechanisms of inhibition for further analysis. One of these small molecules causes the aggregation of actin filaments without binding actin itself, one targets the Aurora B kinase signaling pathway and mis-localizes the Aurora B complex protein INCENP and the third is a potent inhibitor of cytokinesis in mammalian cells. To realize the full potential of these promising small molecules, we propose to identify their cellular targets. We have designed an interdisciplinary approach, combining chemistry, imaging techniques and biochemistry to use the small molecules to probe different aspects of the mechanism of cytokinesis. In addition to being useful tool compounds, our small molecule inhibitors of cytokinesis may also catalyze the development of therapeutic cancer drugs. Many mechanisms underlying cytokinesis, the final step in cell division, remain poorly understood. Failed cell division is causal or contributory in diseases such as cancer. Small molecules that specifically target cytokinesis, the subject of this proposal, are important tools to study the underlying biology of cell division and could catalyze the discovery of therapeutic drugs to treat cancer.